DDS diagnostic probe systems for MDR-TB Principal Investigator, Shafer, David A. PhD Abstract: Tuberculosis (TB) is a primary cause of morbidity and mortality in many areas of the world with about 8 million new cases and 2 million deaths per year. In recent years, there has been a rapid and alarming increase in the incidence and spread of drug resistant TB and the frequency of multi-drug resistant TB (MDR-TB) is increasing worldwide. With most new cases arising from non-compliance or inappropriate treatment of drug susceptible TB, there is a serious need for rapid and precise diagnosis, particularly in the low-resource settings where TB and MDR-TB are prevalent. This translational grant is developing a set of molecular diagnostic probes for MDR-TB based upon new dual-component error-preventing probe technologies that were invented previously by the PI and that enable precise, clear-cut and reliable discrimination of single base mutants associated with resistance. These probes are suitable for end-point and array detection as well as real-time PCR (qPCR). Compared to standard probes for qPCR, these DDS (DNA Detection Switch) probes provide equivalent detection over a wide range of annealing/hybridization temperatures and they can selectively detect a single base mutant and exclude detection of alternate variants at the same site. The program will focus on the design and testing of a comprehensive set of such DDS probes for qPCR that can detect the primary mutants in the rpoB gene that confer Rifampin (RIF) resistance and the key mutant sites in katG and inhA that confer Isoniazid (INH) resistance. Preliminary studies reported here demonstrate we have already achieved absolute single base discrimination with important katG and inhA mutants. We have also demonstrated an additional primer-based probe system called G-Force probes that can be used in conjunction with the internal DDS probes to thereby count the mutant vs. amplicon frequency - a new diagnostic capacity. Initial testing will be done with synthetic genes constructed with the major drug resistant variants. Further testing and blind studies will be conducted with DNA samples from well-characterized clinical isolates and pan- susceptible controls that are being provided by our colleagues at the CDC. The proposed DDS probes for MDR-TB can supplant standard Taqman or Molecular Beacon probes for qPCR that are costly, require careful optimization and are prone to false positives. Our DDS probes are also expected to improve upon the line probe assays of Hain and Innogenetics that typically provide shades of gray vs. absolute single base discrimination and that are frequently ineffective with smear negative samples or HIV co-infected patients. We have also outlined a series of potential secondary diagnostic products for MDR-TB that will follow and build on this Phase I program by translating these new probes to a simple chip format, to new research/diagnostic tools, and to a low-cost, hand- held end-point reader that we have already prototyped and that overcomes the need for real-time PCR instruments. The completion of these short and long range goals should provide valuable new tools for high end research and diagnostics and for clinical management in low resource settings. DDS diagnostic probe systems for MDR-TB Principal Investigator, Shafer, David A. PhD Narrative: This grant is developing novel diagnostic probes for real-time PCR detection of multi-drug resistant tuberculosis (MDR-TB). The development program combines a proven nucleic acid amplification platform with new dual-component error-preventing probe technologies called DDS (DNA detection switch) probes that were invented by the PI and that enable highly precise, clear-cut and reliable discrimination of single base mutants over a wide range of annealing/hybridization temperatures. Standard probes do not provide such absolute single base discrimination and they require narrow temperature control. The program will focus on the design and testing of a comprehensive set of iDDS (internal DDS) probes that can detect the primary mutant sites in the rpoB gene that confer Rifampin (RIF) resistance and key mutant sites in the katG gene and the inhA promoter region that confer resistance to Isoniazid (INH). (RIF and INH are the first line drugs for TB control.) This technology can supplant standard Taqman probes for qPCR which are costly, require careful optimization and are prone to false positives, and it improves upon the line probe assays of Hain and Innogenetics that provide shades of gray vs. absolute single base discrimination and that are frequently ineffective with smear negative samples or HIV co-infected patients. These diagnostic probes are intended for commercial distribution in hospital and clinical labs for testing and monitoring infectious disease and for managing treatment.